For the cell number, the most important is the concentration of the cells in the final staining solution which should be between 1-2 million /ml. 

Hi Mara Esposito,

You say "In the scatter plot (Cell count vs PI) the histogram changes the position along the PI axis between the samples". First of all, are you talking about scatter plot or histogram (cell count vs PI)?

Getting back to your question: As a general rule, we start the experiment with equal number of cells across different samples. However, after your culture treatments (addition of damaging agents or cell cycle blocking reagents), you cannot be sure of still having same number of cells for the PI staining. So, in my opinion, it is not very crucial to keep absolutely same number of cells across samples (I hope you don't have big differences in your cell numbers).

My explanation to your data would be the following.

In the first place, do you know if there is a possibility that your treatments change the cell shape or cell granularity? This will be reflected in your scatter plots. And I am guessing that you have gated this scatter plots to obtain the final histogram plots (cell count vs PI). If this is really the case, the changes in the cell morphology might shift the scatter plot across samples and might not be always included in the gating you define for obtaining the histogram plots.

Hope it helps.

Hi Satish, thank you for your help. I think you got the point about the scatter plot. But I don't know if it could be a problem if the histogram plot changes position along the PI axis across samples of control cells (same cell line, no treated).

Just to add to what Satish and Sherif have mentioned, what also matters is that when you resuspend the cells in the staining solution, you have to make sure that the cells are not clumping up. If this happens, then this can also alter the scatter of the cells as the entire clump will be read as 1 big cell, and so, its granularity in return would alter. 

What is also most important is that when you analyze the cells by FACS, make sure that the number of events that you collect is the same across samples. If you are collecting 10000 events for one of your controls, then keep it the same for all of your other samples. It will then help in applying your gate. 

Also, if you are saying that the histogram is altering between the controls, it would also mean that there could be some issue when you are processing the samples. Do you see a change in cell number or is it a change in granularity? Sometimes over/under-fixation with ethanol affects the cells. Also, sometimes if the RNase treatment is not good, then also you can get some background. If you are coupling PI staining solution with RNase, I would suggest that you try doing RNase treatment initially, followed by addition of PI. 

Hope these give you some insights into resolving the issue.

Good luck

Hi

are you doing experiments on same day? are you sure about the voltage settings and instrument settings are similar when you compare them. cell conc could have an effect if:

cell number is too high causing aggregate formation;

cell number too high causing clogging in machine

if all your parameters in instrument settings are same then this shift can only happen if there is aggregate formation

specially when you mention that you have only control cells. can you share some of your plots?

further it could happen if your fixation method has not worked properly

sorry for the long answer but just wanted to cover all that i could think of.

good luck

puja

Hi Puja,

thank you for your reply. I think from your answer I already got some interesting points that could solve some of my problems! I am doing the same experiments on the same day. 

Thank you again for your help!

For the cell number, the most important is the concentration of the cells in the final staining solution which should be between 1-2 million /ml.